Gut complement induced by the microbiota combats pathogens and spares commensals.

Canonically, the complement system is known for its rapid response to remove microbes in the bloodstream. However, relatively little is known about a functioning complement system on intestinal mucosal surfaces. Herein, we report the local synthesis of complement component 3 (C3) in the gut, primarily by stromal cells. C3 is expressed upon commensal colonization and is regulated by the composition of the microbiota in healthy humans and mice, leading to an individual host's specific luminal C3 levels. The absence of membrane attack complex (MAC) components in the gut ensures that C3 deposition does not result in the lysis of commensals. Pathogen infection triggers the immune system to recruit neutrophils to the infection site for pathogen clearance. Basal C3 levels directly correlate with protection against enteric infection. Our study reveals the gut complement system as an innate immune mechanism acting as a vigilant sentinel that combats pathogens and spares commensals.

Microbiome induced complement synthesized in the gut protects against enteric infections.

Canonically, complement is a serum-based host defense system that protects against systemic microbial invasion. Little is known about the production and function of complement components on mucosal surfaces. Here we show gut complement component 3 (C3), central to complement function, is regulated by the composition of the microbiota in healthy humans and mice, leading to host-specific gut C3 levels. Stromal cells in intestinal lymphoid follicles (LFs) are the predominant source of intestinal C3. During enteric infection with Citrobacter rodentium or enterohemorrhagic Escherichia coli, luminal C3 levels increase significantly and are required for protection. C. rodentium is remarkably more invasive to the gut epithelium of C3-deficient mice than of wild-type mice. In the gut, C3-mediated phagocytosis of C. rodentium functions to clear pathogens. Our study reveals that variations in gut microbiota determine individuals’ intestinal mucosal C3 levels, dominantly produced by LF stromal cells, which directly correlate with protection against enteric infection. Highlights: Gut complement component 3 (C3) is induced by the microbiome in healthy humans and mice at a microbiota-specific level.Gut stromal cells located in intestinal lymphoid follicles are a major source of luminal C3 During enteric infections with Citrobacter rodentium or enterohemorrhagic Escherichia coli, gut luminal C3 levels increase and are required for protection. C. rodentium is significantly more invasive of the gut epithelium in C3-deficient mice when compared to WT mice. In the gut, C3-mediated opsonophagocytosis of C. rodentium functions to clear pathogens.

CD8+ T Cells Require ITK-Mediated TCR Signaling for Migration to the Intestine.

The Tec kinase IL-2-inducible T cell kinase (ITK) regulates the expression of TCR-induced genes. Itk-/- T cell responses are impaired but not absent. ITK inhibition prevented colitis disease progression and impaired T cell migration to the colon in mice. To examine the function of ITK in T cell migration to the intestine, we examined the number of gut T cells in Itk-/- mice and then evaluated their expression of gut-homing receptors. Combined with in vitro murine T cell stimulation and in vivo migration assay using congenic B6 mice, we demonstrated an essential role for ITK in T cell migration to the intestine in mice. Reconstitution of Itk-/- mouse CD8+ T cells with IFN regulatory factor 4 restored gut-homing properties, providing mechanistic insight into the function of ITK-mediated signaling in CD8+ T cell migration to the intestinal mucosa in mice.

The Tec kinase ITK is essential for ILC2 survival and epithelial integrity in the intestine.

Innate lymphoid cells (ILC) are lymphocytes that lack an antigen-specific receptor and are preferentially localized in non-lymphoid tissues, such as mucosal barriers. In these locations ILC respond to tissue perturbations by producing factors that promote tissue repair and improve barrier integrity. We show that mice lacking the Tec kinase ITK have impaired intestinal tissue integrity, and a reduced ability to restore homeostasis after tissue damage. This defect is associated with a substantial loss of Type 2 ILC (ILC2) in the intestinal lamina propria. Adoptive transfer of bone marrow ILC2 precursors confirms a cell-intrinsic role for ITK. Intestinal ILC2 numbers in Itk-/- mice are restored by the administration of IL-2 complexes, also leading to improved intestinal tissue damage repair. Reduced Bcl-2 expression in intestinal Itk-/- ILC2 is also restored to WT levels after IL-2 complex treatment, indicating a tissue-specific role for ITK in ILC2 survival in the intestine.

A Small Molecule Inhibitor of ITK and RLK Impairs Th1 Differentiation and Prevents Colitis Disease Progression.

In T cells, the Tec kinases IL-2-inducible T cell kinase (ITK) and resting lymphocyte kinase (RLK) are activated by TCR stimulation and are required for optimal downstream signaling. Studies of CD4(+) T cells from Itk(-/-) and Itk(-/-)Rlk(-/-) mice have indicated differential roles of ITK and RLK in Th1, Th2, and Th17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice. In this study, we examine the consequences of ITK and RLK inhibition using a highly selective and potent small molecule covalent inhibitor PRN694. In vitro Th polarization experiments indicate that PRN694 is a potent inhibitor of Th1 and Th17 differentiation and cytokine production. Using a T cell adoptive transfer model of colitis, we find that in vivo administration of PRN694 markedly reduces disease progression, T cell infiltration into the intestinal lamina propria, and IFN-γ production by colitogenic CD4(+) T cells. Consistent with these findings, Th1 and Th17 cells differentiated in the presence of PRN694 show reduced P-selectin binding and impaired migration to CXCL11 and CCL20, respectively. Taken together, these data indicate that ITK plus RLK inhibition may have therapeutic potential in Th1-mediated inflammatory diseases.

CD4+VEGFR1(HIGH) T cell as a novel Treg subset regulates inflammatory bowel disease in lymphopenic mice.

Regulatory T cells (Tregs) are a specialized subpopulation of T cells that control the immune response and thereby maintain immune system homeostasis and tolerance to self-antigens. Many subsets of CD4(+) Tregs have been identified, including Foxp3(+), Tr1, Th3, and Foxp3neg iT(R)35 cells. In this study, we identified a new subset of CD4(+)VEGFR1(high) Tregs that have immunosuppressive capacity. CD4(+)VEGFR1high T cells, which constitute approximately 1.0% of CD4(+) T cells, are hyporesponsive to T-cell antigen receptor stimulation. Surface marker and FoxP3 expression analysis revealed that CD4(+)VEGFR1(high) T cells are distinct from known Tregs. CD4(+)VEGFR1(high) T cells suppressed the proliferation of CD4(+)CD25(-) T cell as efficiently as CD4(+)CD25(high) natural Tregs in a contact-independent manner. Furthermore, adoptive transfer of CD4(+)VEGFR1(+) T cells from wild type to RAG-2-deficient C57BL/6 mice inhibited effector T-cell-mediated inflammatory bowel disease. Thus, we report CD4(+) VEGFR1(high) T cells as a novel subset of Tregs that regulate the inflammatory response in the intestinal tract.

Bortezomib can suppress activation of rapamycin-resistant memory T cells without affecting regulatory T-cell viability in non-human primates.

BACKGROUND: Memory T cells specific for donor antigens are currently recognized as a significant barrier for maintaining a successful transplant. Furthermore, it has been shown that commonly used immunosuppressive drugs do not alleviate this memory response. Here, we report that rapamycin allows significant proliferation of memory T cells and bortezomib can abrogate the proliferation of rapamycin-resistant memory T cells when preserving the survival of regulatory T cells. METHODS: Peripheral blood mononuclear cells freshly isolated from non-human primates were stimulated with anti-CD3/CD28 antibodies, and inhibitory and apoptotic effects of rapamycin and bortezomib on memory T-cell proliferation were investigated. The CD95 marker in CD3+ T cells was used for the separate enrichment of memory T cells and naïve T cells. RESULTS: Rapamycin at the level even higher than therapeutic concentration could not suppress the proliferation of a significant proportion of memory T cells. However, the combined administration of bortezomib and rapamycin abrogated the proliferation of rapamycin-resistant memory T cells. Furthermore, bortezomib preserved the survival of preexisting CD4+ FoxP3+ regulatory T cells, while inducing apoptosis of CD4+ FoxP3- conventional T cells. The combined administration of low doses of rapamycin and bortezomib also exerted an additive effect on suppressing T-cell proliferation. Cytokine analysis demonstrated that bortezomib could not only suppress rapamycin-permissive interleukin (IL)-6 production, but also production of interferon (IFN)-gamma, IL-4, and IL-10. CONCLUSIONS: This article provides in vitro data from which immunosuppressive regimens for the effective control of memory T cells in non-human preclinical experiments and in clinical trials are selected.

Soluble mediators from human neural stem cells play a critical role in suppression of T-cell activation and proliferation.

Human neural stem cells (hNSCs) can control inflammation in the central nervous system, although the underlying mechanisms are not understood fully. We investigated the immunomodulatory effect of hNSCs on human T cells and the underlying mechanisms. Culture supernatant from an immortalized hNSC cell line, HB1.F3, which has a therapeutic effect on acute stroke and intracerebral hemorrhage, suppressed the proliferation of allogeneically or mitogenically stimulated human peripheral T cells, including the CD3(+)CD103(+) subpopulation. CFSE labeling and flow cytometry showed that the suppression of proliferation was caused by cell cycle arrest and induction of apoptosis. The lack of significant change in caspase-8 levels and the significant reduction in Bcl-2 expression in the affected T cells suggest that the intrinsic pathway plays a major role in soluble-factor-mediated T-cell apoptosis. The addition of culture supernatant from hNSCs to activated T cells reduced the expression of the activation markers CD69 and CD25 at 24 hr after activation, but at 48 hr only CD69 was down-regulated. A cytometry bead assay showed that the secretion of interleukin (IL)-2 decreased significantly, whereas that of IL-4, IL-10, tumor necrosis factor-alpha, and interferon-gamma increased. These results show that hNSCs can negatively affect human peripheral T cells by suppressing their activation and proliferation through soluble mediators, suggesting that hNSCs have a bystander immunomodulatory effect on T cells.